Coronin 1 modulators for the treatment of autoimmune and lymphoproliferative disorders and mycobacterial infections

ABSTRACT

The invention relates to the treatment of mycobacterial infections, autoimmune disorders, lymphoproliferative disorders and induction of immunosuppression following transplantation using coronin 1 and modulators of coronin 1. Particular modulators of coronin 1 are compounds which inhibit the production of coronin 1 or the formation of active coronin 1 from a coronin 1 precursor, partly or entirely inactivate coronin 1, inhibit concentration of coronin 1 at the site of T cell activation, or inhibit the coronin 1 mediated signaling pathway downstream of the T cell receptor. Examples of such modulators are antibody or antibody fragments, coronin 1 peptide fragments or corresponding phosphopeptides, or anti-sense oligonucleotides, e.g. siRNA or shRNA. The invention further relates to a method of screening for a compound effective in the treatment of mycobacterial infections, autoimmune disorders, lymphoproliferative disorders and induction of immunosuppression following transplantation comprising contacting a candidate compound with coronin 1 or coronin 1 expressing cells, and selecting appropriate compounds.

This application is a continuation of U.S. Ser. No. 12/225,538, filedSep. 24, 2008, now U.S. Pat. No. 8,518,372, which is a national stageapplication of PCT/EP2007/052793, filed Mar. 23, 2007, the entireties ofwhich are incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the treatment of mycobacterial infections,autoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation using modulators of coronin1.

BACKGROUND OF THE INVENTION

T cell homeostasis is central to the ability of vertebrate organisms tomount an effective immune response. Lymphocyte precursors, originatingfrom the bone marrow, home to the thymus, where negative and positiveselection results in the production of CD4 or CD8 single positive Tlymphocytes. From the thymus, single positive T lymphocytes seed theperipheral organs, where they cycle for prolonged times between thesecondary lymphoid organs and the blood in a naïve state. Followinginfection, T cells become activated by dendritic cells within peripherallymph nodes, which induces massive proliferation of so-called effector Tcells. After the infection has been cleared, effector cells have to beeliminated in order to maintain peripheral T cell homeostasis.

The signals that are responsible for the selection, proliferation andsurvival of T cells rely on stimulation of the T cell receptor by majorhistocompatibility complex (MHC) molecules that are present on antigenpresenting molecules. While in the thymus, positive selection selectsthose thymocytes recognizing self-MHC molecules, negative selectionensures the elimination of those T cells that strongly recognizeself-peptides in the context of self-MHC. Together, these selectionprocesses within the thymus ensure the generation of naïve,non-autoreactive T cells for population of peripheral organs.

Once in the periphery, however, T cells need to be maintained in a naïvestate while retaining the capacity to rapidly respond to an infection.Also these processes are regulated through activation of T cellreceptors, both for the induction of T cell proliferation following aninfection as well as for maintaining the naïve T cell population. Whilethe distinction between life and death of a T cell appears to becontrolled by the type of interaction with MHC molecules (A. Singer,Curr Opin Immunol 2002, 14, 207-215, A. Lanzavecchia and F. Sallusto,Curr Opin Immunol 2002, 12, 92-98), the molecular components involved inthis decision are largely unknown.

Coronin 1 is also termed P57 or TACO (for tryptophan aspartatecontaining coat protein (G. Ferrari et al., Cell 1999, 97, 435-447).Coronin 1 is an F-actin interacting protein that is transcribed in allcells of the haematopoeitic lineage. Coronin 1 is a member of the WDrepeat family of coronin proteins that are widely expressed in theeukaryotic kingdom (E. L. de Hostos, Trends Cell Biol 1999, 9, 345-350;J. Gatfield et al., Mol Biol Cell 2005, 16, 2786-2798). Whereas inDictyostelium, which contains a single coronin gene, coronin is involvedin actin-dependent processes such as phagocytosis, cell migration andcytokinesis (E. L. de Hostos et al., EMBO J 1991, 10, 4097-4104; M.Maniak et al., Cell 1995, 83, 915-924), in mammalian cells no biologicalactivity has been assigned to any of the coronin homologues.

Mycobacterium spp. are highly successful pathogens that evade innateimmunity by manipulating the host to ensure long term survival. A roleof TACO (Coronin 1) was suggested for mycobacteria survival withinmacrophage phagosomes (G. Ferrari et al., loc. cit.).

SUMMARY OF THE INVENTION

The present invention relates to a method of treating mycobacterialinfections, autoimmune disorders, lymphoproliferative disorders andinduction of immunosuppression following transplantation comprisingadministering coronin 1 or a modulator of coronin 1, and the use ofcoronin 1 and coronin 1 modulators in said treatment and in themanufacture of medicaments for treating mycobacterial infections,autoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation. Furthermore the inventionrelates to a method of treating mycobacterial infections withcalcineurin modulators such as cyclosporin A and tacrolimus (FK506).

The invention further relates to a method of screening for a compoundeffective in the treatment of mycobacterial infections, autoimmune andlymphoproliferative disorders comprising contacting a candidate compoundwith coronin 1 or a coronin 1 expressing cell, and choosing candidatecompounds which selectively modulate activity of coronin 1 or lead toreduced or enhanced expression of coronin 1. The invention furtherrelates to compounds selected by these methods of screening.

Coronin 1 is essential for survival of naive T cells in the periphery.In the absence of coronin 1, T cells are largely depleted from theperiphery as they undergo apoptosis in the secondary lymphoid organs.Coronin 1 is therefore a specific survival factor for peripheral Tcells. Furthermore, coronin 1 is implicated in the survival ofmycobacteria in macrophages. Modulators of coronin 1 are compoundsuseful to treat mycobacterial infections, autoimmune as well aslymphoproliferative disorders. Coronin 1 modulators influence thecalcineurin pathway. Other calcineurin modulators such as cyclosporin Aand FK506 are likewise useful compounds to treat mycobacterialinfections.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Coronin 1 expression in T lymphocytes

Coronin 1 expression in cells of the haematopoietic lineage. Single cellsuspension prepared from the different organs (BM: Bone Marrow; Th:Thymus; Sp: Spleen; LN: Lymph Nodes) indicated and stained for thelymphocyte markers indicated followed by FACScan analysis.

FIG. 2: Generation of coronin 1 deficient mice

(a) Schematic representation of the genomic organization of the coronin1 wild type (WT) and knock-out (KO) locus. The coronin 1 wild type (WT)locus consists of 11 exons of which exon 1 and 11 encode the 5′- and3′-untranslated regions (UTR), respectively. Exon 2 contains the startcodon. The coronin 1 knock-out (KO) locus was generated by specificallytargeting exon 2 of the coronin 1 gene in mouse embryonic stem (ES)cells. The construct employed comprised of flanking homology regions toallow homologous recombination. After successful integration, exon 2 ofcoronin 1 is replaced by the open reading frame for EGFP which willresult in expression of EGFP as a reporter under the control of coronin1 regulatory elements. The sequence encoding EGFP is followed by aneomycin resistance marker (TK-Hygro-Neo) to allow the selection ofpositive ES cell clones for subsequent blastocyst injection.

(b) Analysis of mice by PCR.

(c) Cell lysates were prepared from the Thymus (Th) and spleen (Sp), andequal protein amounts separated by SDS-PAGE and immunoblotted for thedetection of coronin 1 (left panel) and GFP (right panel).

(d) Wild type (WT, left panels) or coronin 1^(−/−) deficient (rightpanels) thymocytes were allowed to adhere to polylysine coated slides,fixed with paraformaldehyde and permeabilized prior to staining usingphalloidin-568, as well as antibodies against tubulin and coronin 1followed by Alexa-Fluor 568 and -633 labeled secondary antibodies,respectively. BF: Bright Field; A: actin; C1: Coronin 1.

FIG. 3: Analysis of the development of the immune system in wild type(WT) and homozygous (−/−) coronin 1 knock-out mice.

Single cell suspensions of the indicated organs (BM: Bone Marrow; Th:Thymus) were prepared and stained with antibodies directed againstantigens being specifically expressed at the various developmentalstages of the lymphoid and myeloid lineage.

(a) Bone marrow cells were stained for the B-cell lineage marker B220and IgM.

(b) Cells of the myeloid lineage were characterized by expression ofCD11 b and B220.

(c) T-lymphocyte populations were analyzed by CD4 and CD8 staining.

(d) Thymi from WT (+/+) or coronin 1 deficient (−/−) mice were preparedfor histology and stained for CD4 as well as CD8 cells using antibodiescoupled to APC (CD4) or FITC (CD8). Magnification: 10×.

(e) Cell suspensions of the thymus of 6 weeks old homozygous (−/−) orwild type (WT) coronin 1 knock-out mice were analyzed by flow cytometryusing antibodies directed against the indicated thymocyte subsets(CD4/CD8 double-negative (DN), double-positive (DP) and single-positive(SP) thymocytes). Cells were counted using a Neubauer chamber. Subsetspecific cell numbers were calculated by referring the percentage of acertain cell type (determined by flow cytometry) to the total cellcounts. Total (T) and subset specific thymocyte cell counts. Filledbars: wild type, open bars: knock-out. Cc: cell counts. Data aremeans+/−SD of 5 animals (n=5).

FIG. 4: Peripheral lymphocyte populations in WT and coronin 1 deficientmice.

Cell suspensions of spleen (Sp) or inguinal lymph nodes (LN) of 6 weeksold homozygous (−/−) or wild type (WT) coronin 1 knock-out mice wereanalyzed by flow cytometry using antibodies directed against theindicated leukocyte markers. FSC: Forward scatter. Cells were countedusing a Neubauer chamber. Lymphocyte specific cell numbers werecalculated by referring the percentage of a certain cell type(determined by flow cytometry) to the total cell counts

(a) Representative flow cytometry profiles of spleen cell suspensions

(b) Total (T) and lymphocyte specific spleen cell counts. Filled bars:wild type, open bars: knock-out. Data are means+/−SD of 5 animals (n=5).

(c) Representative flow cytometry profiles of inguinal lymph node cellsuspensions.

(d) Total and lymphocyte specific inguinal lymph node cell counts.Filled bars: wild type, open bars: knock-out. Data are means+/−SD of 5animals (n=5).

(e,f) Spleen (e) or inguinal (I-LN) or cervical (C-LN) lymph nodes (f)from WT or coronin 1 deficient (−/−) mice were prepared for histologyand stained for the markers indicated as described in the examples.Magnification: 10×.

FIG. 5: Bone marrow transplantations

Sub-lethally irradiated recipient mice of wild type (a-c) or coronin1^(−/−) (d-f) origin received either a 1:1 mixture of WT (Ly5.1⁺) andcoronin 1^(−/−) (Ly5.2⁺) bone marrow cells (50:50) or bone marrow cellsof either origin only (WT and KO). Single cell suspensions were analyzedby FACS for Ly5.1, Ly5.2 and GFP expression. The percentage of Ly5.1,Ly5.2 and GFP positive cells within gates identifying specificlymphocyte subsets was determined. Depicted are means+/−SD of three micein each group (for d-f, in the case of WT 2 animals were used).Reconstitution of thymus (a,d), spleen (b,e) and lymph nodes (c,f). DP:CD4 and CD8 double positive thymocytes. SP: CD4 or CD8 single positivethymocytes. % pc: % positive cells.

FIG. 6: Homing and apoptosis of T cells in the absence of coronin 1

(a) Cells were isolated from thymus (Th), spleen (Sp) and lymph nodes(LN) of wild type (WT) or coronin 1^(−/−) (−/−, C1^(−/−) mice andstained for CD3 and CD62L. Right panel: percentages of CD62L positiveCD3+ cells. Shown is the mean(+/−SD) from 3 mice. (b) Cells wereisolated from thymus, spleen and lymph nodes of wild type (WT) orcoronin 1−/− (−/−) mice and stained for CD3 and incubated with annexinV-APC and PI. Shown are representative dot plots of CD3⁺ cells analyzedfor annexin V and PI. Right panel: percentages of Annexin V positivecells (AV-P). Shown is the mean(+/−SD) from 2 mice.

FIG. 7: Phenotype of macrophages in the presence and absence of coronin1

(a) Bone marrow derived macrophages from wild type (WT) or coronin 1deficient (C1−/−) mice were lysed in SDS-sample buffer and proteinsseparated by SDS-PAGE followed by immunoblotting for coronin 1 (C1) andvisualization by enhanced chemoluminescence.

(b) Wild type (WT) or coronin 1 deficient (C1−/−) bone marrow derivedmacrophages were seeded on coverslips, fixed and stained for actin (A)and coronin 1 (C1) primary antibodies followed by Alexa fluor488 andAlexa fluor568 conjugated secondary antibodies, respectively. BF=brightfield.

(c) Bone marrow derived macrophages from wild type (WT) or coronin 1deficient (C1−/−) mice were laid on the top of the migration chamber andallowed to migrate towards the bottom half containing human serumactivated zymosan. After 4 h of incubation filters were excised andstained with propidium iodide. Bacteria were counted using fluorescencemicroscopy. For quantitation, ˜125 cells were counted in triplicates.CM=number of cells migrated per field. Results are expressed asmean+/−SD and are a representative of at least two independentexperiments.

FIG. 8: Phagocytosis in the presence and absence of coronin 1

Flow cytometric analysis of receptor mediated phagocytosis by bonemarrow derived macrophages isolated from homozygous coronin 1 knock-out(C1−/−) mice or wildtype (WT) littermates.

(a) To study complement receptor 3 (CR3) mediated uptake adherentmacrophages are incubated with GFP expressing Lactobacillus casei (L.c)that have either been treated with fresh (S) or heat inactivated humanserum (HIS, as control for background uptake). After internalization for30 min at 37° C. the cells were washed to remove adherent bacteria,harvested by scraping and analysed by flow cytometry. Rate of uptake isdetermined as the increase in fluorescence as expressed by the medianfluorescence intensity (MFI).

(b) Fc receptor-mediated uptake is analysed by incubating adherentmacrophages with fluorescent polystyrene beads (B) that have either beencoated with IgG or not (as control for background uptake). Flowcytometric analysis of Fc receptor mediated phagocytosis was performedas described for CR3-mediated uptake.

FIG. 9: Trafficking and survival of mycobacteria in wild type andcoronin 1 deficient macrophages

(a) Intracellular trafficking of mycobacteria. Wild type (WT) or coronin1 deficient (C1−/−) macrophages were incubated with mycobacteria for 1 hfollowed by a 3 h chase, methanol fixed and stained for M. bovis BCG andLAMP1. M=merge.

(b) For quantitation, cells (n=150) were scored for the co-localizationof bacteria with lysosomal marker LAMP1 and represented as percentageco-localization with SD+/− values from at least three independentexperiments. LD=lysosomal delivery.

(c) Macrophages from wild type (WT) or coronin 1 deficient C1−/−) micewere incubated for 3 h with mycobacteria, homogenized, and subjected toorganelle electrophoresis. The distribution of organelle-specificmarkers and the amount of bacteria per fraction were determined asdescribed in the Examples. AU=arbitrary units, FN=fraction number,P=protein, β-hex.=β-hexidin, BCG=M. bovis BCG.

(d) Mycobacterial survival. Wild type (WT) or coronin 1 deficient(C1−/−) macrophages were seeded per well in a 96 well plate andincubated with mycobacteria for 1 h and chased for the indicated times(T in hours). Mycobacterial viability was analyzed as described in theExamples. Error bars shown are from data derived from triplicates. Datashown is representative of at least three independent experiments. Timegiven in hours.

FIG. 10: Intracellular transport and survival of mycobacteria in theabsence and presence of calcineurin inhibitors

(a,b) Bone marrow derived macrophages (10×10⁶ cells) from wild type (WT)or coronin 1 deficient (C1−/−) mice were loaded with calcium specificfluophore Fluo3 as described in the Examples and stimulated with eitherionomycin (a) or M. bovis BCG (b; 1 OD stock in Ringers solution) at 50seconds. The fluorescence emission was monitored in channel FL-1 and isplotted against time in seconds (T,s). RF=relative fluorescence. Thedata shown is representative of at least three independent experiments.Upper curve: Wild type mice. Lower curve: coronin 1 deficient mice.

(c) Bone marrow derived macrophages from both wild type (WT) or coronin1 deficient (C1−/−) mice were allowed to adhere to slides in thepresence of BAPTA-AM and infected with M. bovis BCG for 1 h. The cellswere chased for 3 h at the end of which they were fixed and stained formycobacteria and LAMP1 followed by anti-rabbit Alex fluor 488 andanti-rat Alexa fluor 568 conjugated secondary antibodies, respectively.

(d) For quantitation, cells (n=50) were scored for the co-localizationof bacteria with LAMP1 and represented as percentage co-localizationwith SD+/− values from three independent experiments. L.D.=lysosomaldelivery.

(e) Survival of mycobacteria within wild type (WT) or coronin 1deficient (C1−/−) macrophages in the presence of BAPTA-AM. Macrophageswere infected as described in the legend to FIG. 9 and were chased forthe times (hours) indicated. Survival of the bacteria was analyzed asdescribed in the Examples. The data show representative results obtainedfrom at least three independent experiments.

(f) Bone marrow derived macrophages from wild type (WT) or coronin 1deficient (C1−/−) mice were infected with mycobacteria in the presenceor absence of cycloheximide (CHX) (75 μg/ml) and chased for 3 h at theend of which the cells were fixed with methanol and stained formycobacteria and LAMP1 as described in panel (c). For quantitation,cells (n=50) were scored for the co-localization of bacteria withlysosomal marker LAMP1 and represented as percentage co-localizationwith SD+/− values from three independent experiments. LD=lysosomaldelivery.

(g) Wild type (WT) or coronin 1 deficient (C1−/−) bone marrow derivedmacrophages were infected with M. bovis BCG in the presence ofcyclosporin A (CsA, 0.1 μM) or FK506 (0.5 μM) and chased for 3 h at theend of which the cells were methanol fixed and stained for mycobacteriaand LAMP1 as described in panel (c). For quantitation, cells (n=50) werescored for the co-localization of bacteria with lysosomal marker LAMP1and represented as percentage co-localization with SD+/− values fromthree independent experiments. LD=lysosomal delivery.

(h) Viability of mycobacteria within wild type bone marrow derivedmacrophages in the presence of cyclosporin A (CsA) and FK506.Macrophages were infected as described in the legend to FIG. 9 andchased for the times indicated (hours). Survival of the bacteria wasanalyzed as described in the Examples. The data show representativeresults obtained from at least three independent experiments.

FIG. 11: T cell activation in wild type and coronin 1 deficient T cells.

(a,b) Calcium mobilization upon CD3 (a) or CD3/CD28 (b) stimulation wasmeasured in splenic T-cells by Indo-1 fluorescence and is displayed asthe FL4/FL5 ratio (Relative Calcium Flux, RF). Total splenocytes wereloaded with Indo-1 by incubation with Indo-1 AM, and labeled withanti-CD19-PE and anti-CD11b-PE. Only PE negative cells were analyzed forcalcium mobilization upon CD3/CD28 stimulation. The arrows indicate thetime of addition of the stimulus. The relative calcium flux is expressedas the FL4/FL5 ratio. Shown is one representative out of 3 experimentsusing independent pairs of mice. Less than 5% of the gated cells stainedpositive for annexin V (data not shown).

(c) Interleukin-2 (in pg/ml) production by wild type (filled bars) andcoronin 1−/− (open bars) spleen suspensions following the indicatedstimulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of treating mycobacterialinfections, autoimmune disorders, lymphoproliferative disorders andinduction of immunosuppression following transplantation comprisingadministering coronin 1 or a modulator of coronin 1, and the use ofcoronin 1 and coronin 1 modulators in said treatment and in themanufacture of medicaments for treating mycobacterial infections,autoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation.

Coronin 1 modulators are compounds which downregulate the overallcoronin 1 levels within a T cell. More particularly, coronin 1modulators are compounds which inhibit the production of coronin 1 orthe formation of active coronin 1 from a coronin 1 precursor, partly orentirely inactivate coronin 1, inhibit concentration of coronin 1 at theimmunological T cell synapse, or inhibit the coronin 1 mediatedsignaling pathway downstream of the T cell receptor.

Coronin 1 modulators are furthermore coronin 1 agonists, which reinforcecoronin 1 concentration at the site of T cell stimulation.

Coronin 1 production can be inhibited by anti-senseoligodeoxynucleotides, siRNAs (small interfering RNA) or shRNA (smallhairpin RNA). Prevention of the formation of active coronin 1 can beachieved by targeting coronin 1 precursors. A further example of acompound considered as coronin 1 modulator according to the invention isa binding composition which specifically and/or selectively bindscoronin 1, and which neutralizes coronin 1, i.e. a compound comprisingantigen binding sites of anti-coronin 1 Fabs.

Targeting of coronin 1 can be achieved by the administration ofneutralizing antibodies to coronin 1 or by the administration ofproteins or synthetic compounds, which bind coronin 1, and therebyprevent its natural function in T cells. Alternatively, peptidescomprising parts of coronin 1 can be used as coronin 1 modulatorsaccording to the invention, for example the C-terminal coiled coil, thedifferent segments that make up the seven-bladed propeller domain or thelinker domain (J. Gatfield et al., Mol Biol Cell 2005, 16, 2786).Further examples of coronin 1 modulators according to the invention arecompounds which by binding to coronin 1 interfere with immunological Tcell stimulation.

Modulation of coronin 1 can also be accomplished by gene therapy, forexample by using vectors harboring cDNA, which encodes for proteinsbinding coronin 1 genes, cDNA coding for coronin 1 parts, for examplethe C-terminal coiled coil or the WD repeat domain of coronin 1, or cDNArelated to genes involved in the activation or inactivation ofcoronin 1. Such cDNA and vectors harboring cDNA are likewise consideredcoronin 1 modulators according to the invention.

Anti-coronin 1 antibodies considered to be coronin 1 modulatorsaccording to this invention may be obtained as follows:

Polyclonal antisera are raised against KLH-coupled peptides spanningdifferent regions of the coronin 1 sequence in New Zealand whiterabbits, and antibodies are affinity purified by affinity chromatographyusing the immunizing peptides as ligands. For the generation ofantiserum against recombinantly expressed coronin 1, the coding sequence(full sequence or parts of the sequence) of coronin 1 can be fused tothe GST sequence in the pGEX-4T-1 expression vector, and the fusionprotein can be expressed in E. coli, and purified. Both native andSDS-denatured recombinant coronin 1 can be used for immunization ofrabbits to obtain an anti-coronin 1 antiserum.

Monoclonal antibodies can be generated by immunizing rats (for exampleWistar) with peptides (15-20-mer) and fusion of the spleen and lymphnodes, for example, with SP2/0 myeloma cells. Alternatively, monoclonalantibodies can be prepared in mice (for example Balb/c) followingimmunization and fusion of spleen and lymph nodes with the appropriatemyeloma cell lines, such as P3X63-Ag8.653 (Balb/c).

Both poly- and monoclonal anti-coronin peptides can be generated againstrecombinant protein, fragments thereof or peptide sequences (15-20-mers)produced as described below.

Antibodies (monoclonal and/or polyclonal) are purified and Fab fragmentsare prepared by partial fragmentation, for example by cleavage withpapain or pepsin, which produces two Fab fragments or F(ab)2 fragments,respectively. Also, the DNA that encodes the binding portion ofmonoclonal mouse antibodies can be cloned and merged with human antibodyproducing DNA. Mammalian cell cultures can be used to express this DNAand produce such humanized antibodies. Another approach involves micegenetically engineered for the production of such antibodies.

Preferred coronin 1 peptides useful as coronin 1 modulators according tothe invention are the following peptides derived from human coronin 1:

Amino acid residues 400-416, representing the domain interacting withthe cytoskeleton:

(SEQ ID NO: 1) KSRELRVNRGLDTGRRR

Amino acid residues 430-461 that constitute the coiled coil domain:

(SEQ ID NO: 2) VSRLEEEMRKLQATVQELQKRLDRLEETVQAK (SEQ ID NO: 3)N-terminal domain: MSRQVVRSSKFRHVFGQPAKADQCYE 

Peptides spanning the WD repeats (WD repeats are characterized by a 30to 40 amino acid residue segment bordered by Gly-His (GH) and Trp-Asp(WD) di-peptide residues):

(SEQ ID NO: 4) VCGHTAPVLDIAWCPHNDNVIASGSEDCTVMVWE (SEQ ID NO: 5)LEGHTKRVGIVAWHTTAQNVLLSAGCDNVIMVWD (SEQ ID NO: 6)PEVHPDTIYSVDWSRDGGLICTSCRDKRVRIIE (SEQ ID NO: 7)DRPHEGTRPVRAVFVSEGKILTTGFSRMSERQVALWD (SEQ ID NO: 8)PLSLQELDTSSGVLLPFFDPDTNIVYLCGKGDSSIRYF

Peptides can be synthesized on an automated peptide synthesizer, such asthe Abimed AMS 422 Multiple Peptide Synthesizer, using, for example, thestandard Fmoc chemistry on solid supports (E. Atherton and R. C.Sheppard, Solid Phase peptide synthesis: a practical approach. IRLPress, Oxford, England, 1989).

Alternatively, peptide fragments such as those described above can becloned into the appropriate expression vectors with or without a tag toallow affinity purification, and expressed in mammalian cell culturesystems.

Coronin 1 phospho-peptides are likewise considered coronin 1 modulatorsaccording to the invention. Phospho-peptides can be synthesized asdescribed above, by incorporating the appropriate phospho-amino acidsinstead of the non-phosphorylated amino acid residues during synthesis.For the synthesis of the phospho-peptides, phospho-amino acids derivedfrom serine, threonine or tyrosine, preferably from serine or threonine,are used and 15- or 20-mers prepared. Preferred are phospho-peptides ofthe sequences listed above as coronin 1 peptides, wherein one or more,for example one, two, three, four or five, serine and/or threonineresidues are phosphorylated.

Further considered as coronin 1 modulators are suitable siRNA and shRNA.Sequences that will be used to generate siRNA or shRNA in order to blockcoronin 1 expression are based on the following target sequences, asdesigned according to the rules as, for example, described in C. Sachseet al., Methods Enzymol. 2005, 392, 242-277:

Target sequence AAGTTCCGCCACGTGTTTGGA (SEQ ID NO: 9)Sense strand siRNA: GUUCCGCCACGUGUUUGGA (SEQ ID NO: 10)Antisense strand siRNA: UCCAAACACGUGGCGGAACTarget sequence AAGGCCGACCAGTGCTATGAA (SEQ ID NO: 11)Sense strand siRNA: GGCCGACCAGUGCUAUGAA (SEQ ID NO: 12)Antisense strand siRNA: UUCAUAGCACUGGUCGGCCTarget sequence AAGATGTGCGCGTCTCACAGA (SEQ ID NO: 13)Sense strand siRNA: GAUGUGCGCGUCUCACAGA (SEQ ID NO: 14)Antisense strand siRNA: UCUGUGAGACGCGCACAUCTarget sequence AACCCTAAGTTTGTGGCCCTG (SEQ ID NO: 15)Sense strand siRNA: CCCUAAGUUUGUGGCCCUG (SEQ ID NO: 16)Antisense strand siRNA: CAGGGCCACAAACUUAGGGTarget sequence AAGTTTGTGGCCCTGATCTGT (SEQ ID NO: 17)Sense strand siRNA: GUUUGUGGCCCUGAUCUGU (SEQ ID NO: 18)Antisense strand siRNA: ACAGAUCAGGGCCACAAAC

siRNA duplexes are prepared by standard chemistry. Alternatively, suchsequences may be cloned into suitable vectors such as the pSUPER systemor adeno- and lenti viral based systems to generate short hairpin RNA(shRNA) that is subsequently processed into active siRNA (G. D. Fewelland K. Schmitt, Drug Discov. Today 2006, 11, 975-982).

One aspect of the invention relates to a method of treatingmycobacterial infections, autoimmune disorders, lymphoproliferativedisorders and induction of immunosuppression following transplantationcomprising administering coronin 1 or coronin 1 modulators as definedhereinbefore in a quantity effective against mycobacterial infections,autoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression to a mammal in need thereof, for example to a humanrequiring such treatment. The treatment may be for prophylactic ortherapeutic purposes. For the administration, coronin 1 or the coronin 1modulator is preferably in the form of a pharmaceutical preparationcomprising the coronin 1 or the coronin 1 modulator in chemically pureform and optionally a pharmaceutically acceptable carrier and optionallyadjuvants. Coronin 1 or the coronin 1 modulator is used in an amounteffective against mycobacterial infections, autoimmune andlymphoproliferative disorders. The dosage of the active ingredientdepends upon the species, its age, weight, and individual condition, theindividual pharmacokinetic data, the mode of administration, and whetherthe administration is for prophylactic or therapeutic purposes. In thecase of an individual having a bodyweight of about 70 kg the daily doseadministered is from approximately 1 mg to approximately 500 mg,preferably from approximately 10 mg to approximately 100 mg, of coronin1 or a coronin 1 modulator.

In particular, the compounds of the invention are useful in thetreatment and/or prevention of diseases or disorders mediated bylymphocyte interactions, e.g. in transplantation, such as acute orchronic rejection of cell, tissue or organ allo- or xenografts ordelayed graft function, graft versus host disease, autoimmune diseases,e.g. rheumatoid arthritis, systemic lupus erythematosus, Hashimoto'sthyroidis, multiple sclerosis, myasthenia gravis, diabetes type I or IIand the disorders associated therewith, vasculitis, pernicious anemia,Sjoegren syndrome, uveitis, psoriasis, Graves ophthalmopathy, alopeciaareata and others, allergic diseases, e.g. allergic asthma, atopicdermatitis, allergic rhinitis/conjuctivitis, allergic contactdermatitis, inflammatory diseases optionally with underlying aberrantreactions, e.g. inflammatory bowel disease, Crohn's disease orulcerative colitis, intrinsic asthma, inflammatory lung injury,inflammatory liver injury, inflammatory glomerular injury,atherosclerosis, osteoarthritis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis, cutaneousmanifestations of immunologically-mediated disorders, inflammatory eyediseases, keratoconjuctivitis, myocarditis or hepatitis,ischemia/reperfusion injury, e.g. myocardial infarction, stroke, gutischemia, renal failure or hemorrhage shock, traumatic shock, cancer,e.g. T cell lymphomas or T cell leukamias, infectious diseases, e.g.toxic shock (e.g. superantigen induced), septic shock, adult respiratorydistress syndrome or viral infections, e.g. AIDS, viral hepatitis orchronic bacterial infection, e.g. tuberculosis. Examples of cell, tissueor solid organ transplants include e.g. pancreatic islets, stem cells,bone marrow, corneal tissue, neuronal tissue, heart, lung, combinedheart and lung, kidney, liver, bowel, pancreas, trachea or oesophagus.Furthermore, the compounds of the invention are useful in the treatmentand/or prevention of mycobacterial infections. Mycobacterial infectionsinclude tuberculosis, caused by Mycobacterium tuberculosis, leprosy,caused by Mycobacterium leprae, as well as infections caused byMycobacterium marinum, Mycobacterium bovis, Mycobacterium avium,Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacteriummalmoense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacteriumxenopi, Mycobacterium scrofulaceum—associated with lymphadenitis,Mycobacterium abscessus, Mycobacterium chelonae, Mycobacteriumhaemophilum, and Mycobacterium ulcerans.

Pharmaceutical compositions for enteral administration, such as nasal,buccal, rectal or, especially, oral administration, and for parenteraladministration, such as subcutaneous, intravenous, or intramuscular areespecially preferred. The pharmaceutical compositions comprise fromapproximately 1% to approximately 95% active ingredient, preferably fromapproximately 20% to approximately 90% active ingredient.

For parenteral administration preference is given to the use ofsolutions of coronin 1 or the coronin 1 modulator, and also suspensionsor dispersions, especially isotonic aqueous solutions, dispersions orsuspensions which, for example, can be made up shortly before use. Thepharmaceutical compositions may be sterilized and/or may compriseexcipients, for example preservatives, stabilizers, wetting agentsand/or emulsifiers, solubilizers, viscosity-increasing agents, salts forregulating osmotic pressure and/or buffers and are prepared in a mannerknown per se, for example by means of conventional dissolving andlyophilizing processes.

For oral pharmaceutical preparations suitable carriers are especiallyfillers, such as sugars, for example lactose, saccharose, mannitol orsorbitol, cellulose preparations and/or calcium phosphates, and alsobinders, such as starches, cellulose derivatives and/orpolyvinylpyrrolidone, and/or, if desired, disintegrators, flowconditioners and lubricants, for example stearic acid or salts thereofand/or polyethylene glycol. Tablet cores can be provided with suitable,optionally enteric, coatings. Dyes or pigments may be added to thetablets or tablet coatings, for example for identification purposes orto indicate different doses of active ingredient. Pharmaceuticalcompositions for oral administration also include hard capsulesconsisting of gelatin, and also soft, sealed capsules consisting ofgelatin and a plasticizer, such as glycerol or sorbitol. The capsulesmay contain the active ingredient in the form of granules, or dissolvedor suspended in suitable liquid excipients, such as in oils.

Transdermal application is also considered, for example using atransdermal patch, which allows administration over an extended periodof time, e.g. from one to twenty days.

Another aspect of the invention relates to the use of coronin 1 orcoronin 1 modulators as described hereinbefore in the treatment ofautoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation and in the manufacture ofmedicaments for treating autoimmune disorders, lymphoproliferativedisorders and induction of immunosuppression following transplantation.Such medicaments are manufactured by methods known in the art,especially by conventional mixing, coating, granulating, dissolving orlyophilizing.

Coronin 1 or the coronin 1 modulator can be administered alone or incombination with one or more other therapeutic agents, possiblecombination therapy taking the form of fixed combinations of coronin 1or the coronin 1 modulator and one or more other therapeutic agentsknown in the treatment of mycobacterial infections, autoimmunedisorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation, the administration beingstaggered or given independently of one another, or being in the form ofa fixed combination.

The invention further relates to a method of screening for a compoundeffective in the treatment of mycobacterial infections, autoimmunedisorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation comprising contacting acandidate compound with coronin 1 or a coronin 1 expressing cell, andchoosing candidate compounds which selectively modulate activity ofcoronin 1 or lead to reduced or enhanced expression of coronin 1. Theinvention further relates to compounds selected by these methods ofscreening.

Inhibitors of coronin 1 activity are identified by contacting coronin 1with a candidate compound. A control assay with coronin 1 in the absenceof the candidate compound is run in parallel. A decrease in activity inthe presence of the candidate compound compared to the level in theabsence of the compound indicates that the compound is a coronin 1inhibitor.

The present invention likewise relates to a method of treatingmycobacterial infections, comprising administering calcineurinmodulators such as cyclosporin A and tacrolimus (FK506), and the use ofcalcineurin modulators such as cyclosporin A and tacrolimus (FK506) insaid treatment and in the manufacture of medicaments for treatingmycobacterial infections.

Concepts and Evidence Behind the Invention

Coronin 1 is important for survival of T lymphocytes in the periphery;in the absence of coronin 1, naïve T cells undergo apoptosis and arefully depleted from secondary lymphoid organs. In contrast,developmental processes in the thymus are not compromised in the absenceof coronin 1. Coronin 1 specifically functions in the survival ofperipheral T cells without affecting T cell development in the thymus.

The reason for T cells to become deleted from the periphery is becausecoronin 1 is essential to allow Ca²⁺ signaling following T cell receptortriggering (FIGS. 10 a and b).

The peripheral T cell population is maintained at near constant levels;maintenance of naïve T cells in the periphery is dependent on theappropriate stimulation of T cells through their T cell receptors.Indeed, in hosts that lack peripheral MHC class I or class IIexpression, T cells are unable to survive because of the absence ofTCR-mediated T cell stimulation (J. Kirberg et al., J. Exp. Med. 1997,186, 1269-1275). Also, induction of T cell receptor deletion results inthe disappearance of peripheral T cells (N. Labrecque et al., Immunity2001, 15, 71-82) showing that T cells need input signals through T cellreceptor triggering to support their survival in the periphery.

Triggering of the T cell receptor results in the activation of Ca²⁺dependent signaling pathways, that in turn activates a diverse array oftranscription factors (E. M. Gallo et al., Nat. Immunol. 2006, 7,25-32). Activation of such Ca²⁺ dependent pathways occurs followinginflux of Ca²⁺ into the cytosol at two stages. First, T cell receptortriggering stimulates Ca²⁺ release from the intracellular endoplasmicreticulum stores which results in a rapid and transient increase incytosolic Ca²⁺ concentration (M. M. Winslow et al., Curr. Opin. Immunol.2003, 15, 299-307). Second, depletion of these intracellular storesresults in the opening of plasma membrane Ca²⁺ channels, the so-calledstore operated channels (SOC) or Ca²⁺ release-activated Ca2+ (CRAC)channels.

In this invention it is shown that coronin 1 controls TCR-dependentcytosolic Ca²⁺ mobilization from intracellular stores and plasmamembrane CRAC channels, thereby regulating the survival of naïve T cellsin the periphery (FIG. 11). In the absence of coronin 1, Ca²⁺mobilization does not occur, and, as a consequence, translocation of theT cell specific transcription factor NFAT is not induced resulting indefective interleukin-2 production and T cell death. Coronin 1 is aspecific survival factor for peripheral T cells, and, as a consequence,compounds inhibiting or modulating coronin 1 are useful in the treatmentof autoimmune disorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation. In fact, the mostsuccessful current immunosuppressants rely on the abrogation of Ca²⁺signaling, as exemplified by the successful treatment of autoimmunedisorders, lymphoproliferative disorders and induction ofimmunosuppression following transplantation using cyclosporin A or FK506(tacrolimus), both of which block NFAT translocation and IL-2 productionby inhibiting the activation of the Ca²⁺ dependent phosphatasecalcineurin.

Autoimmunity is a disorder that is caused by the presence ofpredominantly T cells that become activated against self tissue in theperipheral organs (for examples diabetes or multiple sclerosis).Similarly, T cells play a central role in the specific immune responseof allograft rejection. Strategies to prevent T cell activation,proliferation or effector function are thus all potentially useful forimmunosuppression for the treatment of autoimmunity and transplantation(K. L. Hardinger et al., Pharmacotherapy 2004, 24, 1159-1176).

The presence of T cells and their activation in the peripheral tissuesthat cause autoimmunity or transplant rejection depend on the productionof the essential cytokine interleukin-2 by T cells. Stimulation of the Tcell receptor that results in T cell activation and interleukin-2production occurs through the presentation of self antigens in the caseof autoimmune diseases, and foreign antigens in the case of transplantrejection by the molecules of the Major Histocompatibility Complex(MHC).

Triggering of the T cell receptor results, via tyrosine phosphorylationof adaptor molecules, to influx of calcium from intracellular andextracellular stores that is dependent on coronin 1 (FIG. 11). Thecoronin 1-dependent increase of calcium activates the Ca²⁺ dependentphosphatase calcineurin, that, via dephosphorylation of the T celltranscription factor Nuclear Factor of Activated T cells (NFAT) resultsin interleukin-2 gene transcription and interleukin-2 production. In theabsence of coronin 1, calcium mobilization cannot occur and thereforeinterleukin-2 is not produced resulting in T cell depletion.

The phenotype of coronin 1−/− mice with respect to the absence of Tcells from the periphery is reminiscent of that of mice containing adeletion of genes involved in lymphocyte migration (Y. Fukui et al.,Nature 2001, 412, 826-831; M. Matloubian et al., Nature 2004, 427,355-360). However, the finding that coronin 1−/− mice do not display anaberrant actin or tubulin cytoskeleton nor accumulated single positive Tcells in the thymus is inconsistent with such a migration defect.Lymphocyte homing is not compromised which is consistent with the normalacquisition of the lymphocyte homing receptor CD62L on coronin 1deficient thymocytes.

Rather than functioning in lymphocyte homing and migration, coronin 1may modulate the strength of signaling downstream of the T cellreceptor. This is suggested by the fully normal development within thethymus of coronin 1−/− mice. Also, the increased apoptosis of coronin 1deficient mice suggests a lack of the appropriate stimulus that isnormally required to allow survival of T cells in the periphery (S.Takeda et al., Immunity 1996, 5, 217-228). Interestingly, coronin 1binds to cellular membranes in a cholesterol-dependent manner (J.Gatfield and J. Pieters, Science 2000, 288, 1647-1650) and its locationat the immunological synapse suggest an important function during T cellsignaling. While additional mechanisms must exist to differentiatethymus-derived signals from those that T cells receive in the periphery,coronin 1 is important for signal transmission in the periphery only.

Coronin 1 in T Cells

Coronin 1 is an F-actin interacting protein that is transcribed in allcells of the haematopoietic lineage (G. Ferrari et al., Cell 1999, 97,435-447). Analysis of the different haematopoietic cell types usingFACScan for the expression of coronin 1 reveals that T cells expresscoronin 1 most abundantly (FIG. 1), with a more moderate expression in Bcells and macrophages.

Coronin 1 Deficient Mice

To analyze a role for coronin 1 during T cell stimulation, mice lackingcoronin 1 were generated by homologous recombination in embryonic stemcells (FIG. 2 a). To be able to analyze the in vivo pattern of coronin 1expression, exon 1 was replaced with the enhanced green fluorescentprotein coding region (FIG. 2 a,b). Coronin 1−/− mice were born at theexpected mendelian ratio, were fertile and appeared to be healthy. Asexpected, thymus and spleen from coronin 1−/− animals lacked anydetectable coronin 1 expression, confirming ablation of coronin 1 inlymphocytes (FIG. 2 c). The morphological appearance as well as theF-actin and tubulin cytoskeleton of the B cells, T cells and macrophagesin coronin 1−/− deficient animals was indistinguishable from leukocytesthat were isolated from wild type littermates (FIG. 2 d), suggesting nogross defects in leukocyte cytoskeletal structure and/or morphology.

A contribution of coronin 1 was investigated in lymphocyte development.To that end, bone marrow and thymus from wild type and coronin 1deficient animals were analyzed for the presence of the differenthaematopoeitic lineages. Both in the bone marrow as well as in thethymus, lymphocytes appeared to be normal, with no differences in eitherrelative or absolute numbers of lymphocytes present (FIG. 3 a-c). In thethymus of coronin 1−/− mice, relative subsets of double negative, doublepositive and single positive T cells were within the normal range ofcellularity, although there was a slight but consistent increase in thetotal number of double positives (FIG. 3 e). In addition, histologicalanalysis of thymi from wild type and coronin 1−/− mice revealed nodifferences in the overall organization as well in the proportion of Tcell subsets (FIG. 3 d). Therefore it is concluded that lymphocytedevelopment into CD4 and CD8 single positive T cells occurs normally inthe absence of coronin 1.

T Cell Populations in the Periphery

In contrast to normal thymic population and architecture, the analysisof the lymphocyte populations in the periphery reveals grossabnormalities. Analysis of tailblood of coronin 1 deficient animalsreveals a drastic reduction in the number of leukocytes, while all othercell types were present in equal numbers (Tables I and II).

TABLE I Peripheral Blood Composition (ADVIA Analysis) Wild type Coronin1 ^(−/−) Leukocytes (×10⁹/l) 15.64 ± 5.77  4.77 ± 1.4  Erythrocytes(×10¹²/l) 10.4 ± 2   10.12 ± 1.47  Hemoglobin (g/l) 161.6 ± 22.53 164.05± 28.9  Thrombocytes (×10⁹/l) 1419.88 ± 265    1101.5 ± 265  Reticulocytes (×10⁹/l) 413.54 ± 116.1  393.4 ± 138.1

TABLE II Peripheral Blood Leukocyte Composition (FACscan Analysis) Wildtype Coronin 1 ^(−/−) CD4⁺ (×10⁴/ml) 76.6 ± 12.7 4.4 ± 2.4 CD8+(×10⁴/ml) 51.2 ± 9.7  3.6 ± 2   CD19+ (×10⁴/ml) 168.5 ± 48.9  48.6 ±16.9 CD11b+ (×10⁴/ml) 64.7 ± 25.4 25.8 ± 13  

Further analysis of the blood reveals that the reduction in leukocytenumbers is largely due to the absence of T lymphocytes. The absence ofperipheral T cells in coronin 1−/− deficient animals was confirmed bythe analysis of spleen and lymph nodes. The spleen of coronin 1deficient mice contained ˜50% of either CD4 or CD8 positive T cellsrelative to wild type littermates, while CD19 as well as CD11b positivecells were only slightly depressed (FIG. 4 a,b). The lymph nodes ofcoronin 1 deficient mice were however almost completely devoid of Tcells when compared to wild type animals (FIG. 4 c,d). Interestingly,the depletion of T cells was most significant in the inguinal lymphnodes, with a 20-fold difference in single positive T cell numbers,whereas T cells within cervical lymph nodes of coronin −/− mice werereduced (FIG. 4 f). Histological analysis revealed that T cells werelargely depleted from the spleen periarteriolar lymphoid sheets, whichwere instead filled up with B cells. Although the lymph nodes fromcoronin 1 deficient mice had only 1/10 of the size of their wild typelittermates, the overall architecture was similar to the architecture ofwild type littermates (FIG. 4 e).

Reconstitution of Lymphoid Organs in Wild type and Coronin 1^(−/−) mixedBone Marrow Chimeras

To analyze whether the specific depletion of peripheral T cells wasreproduced after transplantation of coronin 1^(−/−) bone marrow stemcells in irradiated wild type recipient mice, bone marrow cells ofcoronin 1^(+/+) (CD45 allele=Ly5.1⁺) and coronin 1^(−/−) (Ly5.2⁺) originseparately or in a 50%:50% mixture were transferred into sub-lethallyirradiated six weeks old C57BU6 mice (of Ly5.2 origin). Afterreconstitution, both the thymus as well as the peripheral B cellcompartments were reconstituted in an approximate 50:50 ratio (FIG. 5a,b) reflecting the capacity of coronin 1^(−/−) cells to reconstitutethe thymus as well as the B cell compartment. In contrast, theT-lymphocyte populations in the spleen were largely derived from wildtype cells (FIG. 5 b). For lymph nodes, T cells derived from coronin1^(−/−) bone marrow were barely detectible, as judged by the GFPstaining, indicating that coronin 1 deficient cells were unable toreconstitute the peripheral T-cell compartments (FIG. 5 b,c). Similarly,while wild type bone marrow stem cells were able to fully reconstitutethe lymphoid system of coronin 1 deficient animals, after transfer ofstem cells from coronin 1 deficient mice the resulting animals showed asimilar strong defect in the peripheral lymphoid organs as observed incoronin 1 deficient animals (FIG. 5 d-f). These results thereforefurther corroborate the finding that in the absence of coronin 1T-lymphocytes fail to appear or to be maintained in the periphery.

T cell Survival in the Absence of Coronin 1

The absence of thymocytes from the peripheral lymphoid organs could bedue to a failure to properly develop in the thymus, a deficiency in Tcell homing, or the inability of T cells to survive in the periphery.Since both the cellularity as well as the organization of the thymus inwild type and coronin 1 deficient animals is normal, coronin 1 isunlikely to play a role during T cell development. T cell homing islargely regulated through CD62L (L-selectin), and failure to generateCD62L positive cells in the thymus could be responsible for peripheral Tcell depletion. However, as shown in FIG. 6 a, the percentages of CD62Lsingle positive cells present in the thymus is identical in the presenceand absence of coronin 1, suggesting that T cell homing is notcompromised in the absence of coronin 1.

To analyze whether the depletion of peripheral T cells occurs throughenhanced apoptosis, lymphocytes from thymus, spleen and lymph nodes wereanalyzed for the expression of annexin V and propidium iodide staining,as markers for apoptosis and necrosis, respectively. Staining ofthymocytes revealed identical annexin V and PI positive cellpopulations, confirming no defects in thymocyte development. Incontrast, both within the spleen and lymph nodes the percentage of cellsundergoing apopotosis were increased up to 4-fold. In the absence ofcoronin 1, survival of T cells in the periphery is severely compromisedresulting in the induction of apoptosis

Coronin 1 in Macrophages

Bone marrow derived macrophages isolated from coronin 1 deficient miceshowed the absence of coronin 1 by immunoblotting as well asimmunostaining on fixed cells, whereas the F-actin and tubulincytoskeleton was not affected (FIG. 7 a,b). In addition, motility ofmacrophages from either wild type or coronin 1 deficient macrophages wasfound to be similar as analyzed by Transwell assay (FIG. 7 c). Togetherthese results suggest that coronin 1 does not modulate the macrophageF-actin cytoskeleton in resting or activated conditions.

To directly analyze a role for coronin 1 in phagocytosis, bone marrowmacrophages derived from either coronin 1^(−/−) mice or wild typelittermates were incubated with human serum treated GFP expressingLactobacillus casei or IgG coated fluorescent polystyrene beads for 30min at 37° C. Both wild type as well as coronin 1^(−/−) macrophages werefound to have a similar capacity to take up cargo by either complementreceptor or Fc receptor-mediated phagocytosis (FIG. 8 a,b). Similarly,uptake of latex beads, E. coli as well as Salmonella was found to beidentical in macrophages derived from wild type or coronin 1 deficientmice. To analyze a role for coronin 1 in macropinocytosis, wild type orcoronin 1 deficient macrophages were incubated with FITC-labeled dextranrevealing an identical capacity for macropinocytosis in the presence orabsence of coronin 1. Together these data firmly establish that coronin1 is not essential for phagocytosis or macropinocytosis in macrophages.

In both wild type as well as in coronin 1 deficient macrophages similarnumbers of mycobacteria are internalized. However, analysis of theintracellular localization of mycobacteria internalized for 1 hourfollowed by a 2 hour chase showed that while in wild type macrophages,as expected, mycobacteria were predominantly retained in non-lysosomalphagosomes, in macrophages lacking coronin 1 mycobacteria were largelypresent in LAMP positive vacuoles (FIG. 9 a,b). In addition, subcellularfractionation by organelle electrophoresis independently confirmedlysosomal transfer of mycobacteria in coronin 1 deficient macrophages:while in wild type macrophages, mycobacteria were largely retained innon-lysosomal fractions, in coronin 1 deficient macrophages the majorityof internalized mycobacteria co-localized with lysosomal organelles(FIG. 9 c). Finally, whereas wild type mycobacteria survived readilywithin macrophages, in the absence of coronin 1 mycobacteria wererapidly killed (FIG. 9 d). Together these results demonstrate thatcoronin 1 prevented lysosomal delivery and mediated intracellularsurvival of mycobacteria.

Macrophages from wild type or coronin 1 deficient animals were loadedwith the calcium indicator Fluo-3, and cytosolic Ca2+ influx wasstimulated using ionomycin. While in macrophages derived from wild typemice the addition of ionomycin was accompanied with a flux of cytosolicCa²⁺, in the absence of coronin 1 no Ca²⁺ flux was observed uponstimulation, suggesting coronin 1 deficient macrophages, as is the casein T cells, fail to mobilize Ca²⁺ upon stimulation (FIG. 10 a).Similarly, when Fluo-3 loaded wild type macrophages were allowed tointernalize mycobacteria, this internalization was accompanied withmobilization of cytosolic Ca²⁺ in wild type macrophages. In contrast, inmacrophages lacking coronin 1, no Ca²⁺ flux was observed followinginternalization of mycobacteria (FIG. 10 b).

To analyze whether the coronin 1 mediated Ca²⁺ influx was responsiblefor the block in lysosomal delivery and survival of mycobacteria,intracellular calcium was chelated before and during the internalizationof mycobacteria in macrophages followed by analysis of subcellulardelivery and mycobacterial survival. When intracellular calcium waschelated using the cell permeable chelator BAPTA-AM, virtually allmycobacteria were transported to lysosomes and killed, to a similardegree as in coronin 1 deficient macrophages (FIG. 10 c-e), suggestingthat the coronin 1 mediated raise in cytosolic Ca²⁺ was responsible forblocking phagosome-lysosome fusion and intracellular killing of themycobacteria.

To investigate whether the coronin 1 dependent phagosomal retention andsurvival of mycobacteria was dependent on gene transcription,mycobacteria were internalized in the presence of the protein synthesisinhibitor cyclohexamide. While cyclohexamide efficiently blocked proteinsynthesis without compromising macrophage viability, the presence ofcycloheximide did not result in alteration of the intracellular routingor survival of mycobacteria (FIG. 10 f), indicating that the coronin 1mediated block of phagosome-lysosome fusion did not occur as a result ofinduction of translation due to the activation of transcription factors.

To analyze whether coronin 1 acts through activation of calcineurin toprevent lysosomal delivery of mycobacteria, the calcineurin inhibitorscyclosporin A and FK506 were analyzed for their ability to inducelysosomal delivery and killing of mycobacteria. Both inhibitors induceda complete relocation of mycobacteria to lysosomes (FIG. 10 g).Furthermore, mycobacteria were unable to survive within macrophages whencalcineurin was blocked by either cyclosporin A or FK506 whereasbacterial proliferation was identical in the presence or absence ofcalcineurin inhibitors (FIG. 10 h). Together these results show thatcoronin 1 dependent calcineurin activation is essential to allowintracellular survival of mycobacteria within macrophages.

Examples

Generation of Coronin 1 Deficient Mice

A targeting vector was constructed to replace the entire exon 2 ofcoronin 1 by the coding sequence of the enhanced green fluorescentprotein (EGFP) followed by a neomycin resistance cassette. The codingregion for EGFP was put in frame with the original start codon of thecoronin 1 locus leading to expression of EGFP under the control ofcoronin 1 regulatory elements.

Genomic DNA of mouse embryonic stem cells (129/OIa,TMCF, Biozentrum,Basel, Switzerland) served as a template to amplify by PCR 1.8 kb and2.7 kb flanking homology regions 5′ and 3′, respectively, of the startcodon for coronin 1. Primers included appropriate restriction sites forfurther subcloning and have the following sequence: forward primer (5′flanking region): 5′-GCT TAG CGG CCG CGT CAG CAT CTG TTC GGG GG-3′ (SEQID NO:19); reverse primer (5′ flanking homology region): 5′-CGC AGA ATTCCG CCC ATG GGG CTC ATC CTG AAG GAT ACA G-3′ (SEQ ID NO:20); forwardprimer (3′ flanking region): 5′-GTG GCA CAC GCC TTT AAT CT-3′ (SEQ IDNO:21); reverse primer (3′ flanking region): 5′-GCC ATC GCA GAG TGT TGATA-3′ (SEQ ID NO:22). The coding region for EGFP was PCR amplified frompEGFP-N2 (Clontech) using the following primers: forward primer: 5′-CGAATT CTG CAG TCG ACG GTA CCG-3′ (SEQ ID NO:23) and reverse primer: 5′-CGCAGA ATT CGC CTC GAG TTT ACT TGT ACA GCT CGT CC-3′ (SEQ ID NO:24). Thetargeting vector was constructed by first subcloning the 5′-flankinghomology region via NotI and EcoRI into pBluescript SK+ (Stratagene).Employing NcoI and EcoRI sites the EGFP cassette was inserted 3′ of the5′-flanking homology region. The 3′-flanking homology region was thenadded 3′ of EGFP by XhoI and EcoRI sites. Via an XhoI site, a SV40polyAsignal and the neomycin resistance cassette (pA-lox-TK-Neo-lox, S.Hippenmeyer et al., PLoS Biol 2005, 3, e159) was inserted between EGFPand the 3′-flanking homology region. The ClaI linearized targetingconstruct was electroporated into mouse embryonic stem cells (strain129/Sv) and clones were selected with G418. Correctly targeted ES cellclones were identified by Southern blot and then microinjected intoC57/BI6xBDF1 blastocysts (RCC, Füllinsdorf, Switzerland). The malechimeras obtained were crossed with C57BU6 females. Transgenic offspringwas genotyped by Southern blot to confirm the disruption of thewild-type coronin 1 allele. Mice that were heterozygous for the mutantallele were crossed to generate homozygous coronin 1 deficient mice.Routine PCR screening to specifically detect the mutant allele by theappearance of a 500 bp product was performed with the following primers:forward: 5′-CTG TTG TAG GGG CTG ATG GT-3′ (SEQ ID NO:25); reverse:5′-CTT CAT GTG GTC GGG GTA G-3′ (SEQ ID NO:26). The wild type allele isdetected by PCR amplifying a 400 bp product with the primers: forward:5′-CTG TTG TAG GGG CTG ATG GT-3′ (SEQ ID NO:27) and reverse: 5′-CAC TGGCCT CAC AGA TCA GA-3′ (SEQ ID NO:28).

Biochemical Methods

Cell homogenization, lysis and immunoblotting was performed as described(G. Ferrari et al., Cell 1999, 97, 435-447). Polyclonal anti-coronin 1serum has been described before (J. Gatfield et al., Mol Biol Cell 2005,16, 2786-98). Monoclonal anti-GFP was purchased from Roche (monoclonalmouse IgG₁, clones 7.1 and 13.1).

For immunoblotting, equal number of cells from both wild type or coronin1 deficient bone marrow derived macrophages were lysed in Laemmli samplebuffer and proteins separated by SDS-PAGE (10%) and transferred on anitrocellulose membrane. The membrane was probed with anti-coronin 1antiserum (1:1000 dilution) followed by goat anti-rabbit HRP secondaryantibody and developed by enhanced chemoluminescence. For subcellularfractionation, macrophages were homogenized using a ball bearinghomogenizer (EMBL, Heidelberg, Germany), and the post-nuclearsupernatant was processed for organelle electrophoresis. Bacteria weredetected after sedimentation of the different fractions followed byfixation (PFA) and staining with propidium iodide or using acid faststaining (Becton Dickinson).

Immunofluorescence Microscopy

Cells were adhered on poly-L-lysine coated 10 well Teflon-coated glassslides (Polysciences) for 20 minutes on ice. After fixation (10 min, 3%paraformaldehyde in PBS, 37° C.) and permeabilization in 0.1% saponin/2%BSA in PBS, cells were incubated for 30 min with primary antibodies(anti-coronin 1 antiserum, 1:1000; anti-tubulin, IgG₁ ascites clone E7,1:4000). Following washing (3×0.1% saponin/2% BSA in PBS),phalloidin-AlexaFluor568 (Molecular Probes) and secondary antibodies(goat-anti-mouse AlexaFluor568, goat-anti-rabbit AlexaFluor633;Molecular Probes) were applied for 30 min at 1:200 dilutions. Slideswere washed 3× with 0.1% saponin/2% BSA in PBS and 3× with PBS andmounted using Fluoroguard antifade mounting medium (BioRad) and analyzedusing the confocal laser scanning microscope LSM510 Meta (Zeiss) and thecorresponding software.

For macrophage staining, 5×10⁴ bone marrow derived macrophage cells wereseeded on a three well micro slide (Polysciences) and allowed to adherefor 2 h at 37° C. and 5% CO₂. 200 μl of Mycobacterium bovis BCGsuspension, previously washed three times in fresh BMM medium (DMEMsupplemented with 30% L292 culture supernatant, 10% FCS, 2 mM glutamine,0.5 mM 2-mercaptoethanol) and re-suspended to a final OD of 0.2, wasreplaced gently over the adhered cells and allowed to infect themacrophages for 1 h at 37° C. and 5% CO₂. Non-internalized bacteria wereremoved by three washes with medium, and infected cells were chased foran additional 3 h at the end of which the cells were fixed with methanoland stained for mycobacteria and LAMP1 using rabbit anti Mycobacteriumbovis polyclonal antibody (Dako), and rat anti LAMP1 monoclonal antibody1 D4B, respectively. In experiments involving calcineurin inhibitors(cyclosporin A (0.1 μM) and FK506 (0.5 μM)), calcium chelators (BAPTA-AM(1 μM)) or cycloheximide (75 μg/ml) the reagents were added at the timeof seeding the macrophages.

Flow Cytometric Analysis and Blood Counts

Cell counts were determined for single cell suspensions of the indicatedorgans using a Neubauer chamber. Flow cytometry was carried out bystaining the cells with the relevant monoclonal antibodies at saturatingconcentrations in PBS/2% FCS and analyzing them on a FACSCalibur(Becton-Dickinson). The following monoclonal antibodies and secondaryreagents were obtained from BD Pharmingen: PE-labelled α-CD19 (clone1D3), PE-labelled α-B220 (clone RA3-6B2), PE-labelled α-CD3 (clone145-2C11), PE-labelled α-CD11b (clone M1/70), PECy7-labelled α-CD4(clone RM4-5), APC-labelled α-CD8a (53-6.7), APC-labelled α-CD62L (cloneMEL-14) and PECy7-labelled Streptavidin.

PE-labelled α-CD8a (clone 53-6.7) was obtained from eBioScience.Biotin-labelled antibodies against IgM (clone M41) and CD4 (clone RM4-5)were produced and labelled according to standard techniques.APC-labelled Annexin V (BD Pharmingen) was used according to themanufacturers protocol. Peripheral blood was obtained by tail bleedingand cell counts as well as the hemoglobin concentration were determinedon an ADVIA hematology system (Bayer). For flow cytometry, peripheralblood was subjected to erythrocyte lysis prior to staining and analysison a FACSCalibur.

Immunohistology

Organs were embedded in OCT medium (Sakura Finetek), snap-frozen and 5μm sections were cut with a cryostat. Sections were air-dried,acetone-fixed for 8 min and stored at −70° C. Sections were thenrehydrated and blocked in PBS/2% BSA with 0.1% NaN₃ and 220 μg/ml mouseIgG (Jackson ImmunoResearch Laboratories). Antibodies, diluted in PBS/2%BSA, were added directly onto the sections and incubated for 60 min atroom temperature in a wet chamber. Antibodies used were: Allophycocyanin(APC)-labelled anti-mouse CD4 (rat IgG2a, clone RM4-5) (CaltagLaboratories), fluorescein isothiocyanate (FITC)-labelled anti-mouse CD8(rat IgG2a, clone 53-6.7) (BD Biosciences), fluorescein isothiocyanate(FITC)-labelled anti-mouse B220/CD45R (rat IgG2a, clone RA3-6B2) (BDBiosciences), R-Phycoerythrin (PE)-labelled anti-mouse Thy1.2/CD90.2(mouse IgG2b, clone 5a-8) (Caltag Laboratories) and R-Phycoerythrin(PE)-labelled anti-mouse I-A^(b) (mouse IgG2a, clone AF6-120.1) (BDBiosciences). Sections were counterstained with dapi (Serva) for 5 min.After washing the sections were mounted in Fluoromount (SouthernBiotechnology Associates). Images were taken on a Zeiss Axioskop withORCA ER camera (Hamamatsu) and images were processed by using OPENLABsoftware (Improvision, Coventry, U.K.).

Stimulation of T-Lymphocytes

Polystyrene beads (3 or 6 μm diameter; Polysciences) were coated withanti-CD3 (50 μg/ml, clone 145-2C11, BD Pharmingen) antibody in PBS for 1hr at 37° C. on a shaking thermoblock. Total thymocytes (9×10⁵) orFicoll purified splenic lymphocytes (9×10⁵) were mixed with 1.5×10⁶coated beads in IMDM/5% FCS/2 mM glutamine and gently pelleted (250×g, 2min., 4° C.). Following incubation at 37° C. for 30 min. cells wereresuspended in PBS using blunt-ended pipette tips, adhered topoly-L-lysine coated slides and stained for confocal microscopy.

Mixed Bone Marrow Chimeras

Donor bone marrow cells were isolated from either Ly 5.2 positivehomozygous coronin 1 knock-out mice or from Ly5.1 positive C57BU6 mice.Bone marrow cells were harvested by flushing isolated femurs and tibiaswith a syringe and needle using serum-free IMDM. Six weeks old recipientC57BU6 mice (Ly5.2 positive) were irradiated with 950 rad and receivedthen 2×10⁶ bone marrow cells comprising of a 1:1 mixture of Ly5.2⁺coronin 1−/− and Ly5.1⁺ C57BL/6 cells. As controls irradiated recipientsalso received 2×10⁶ of either Ly5.2⁺ coronin 1−/− or Ly5.1⁺ C57BL/6cells alone. Composition and origin of lymphoid cells in chimeric micewas analyzed six weeks later by flow cytometry employing antibodystainings specifically detecting the Ly5.1 or Ly5.2 markers.

Cell Migration

Migration assays were performed in Costar24 well migration chambers withpore size of 8 μm. Cells were laid on the top of the chamber and allowedto migrate towards the bottom half containing chemo attractant (humanserum activated zymosan). After 4 h of incubation filters were excisedand stained with propidium iodide. Cells were counted using fluorescencemicroscopy.

Mycobacterial Survival

5×10⁴ bone marrow derived macrophages in BMM media were seeded per wellin a 96 well plate (Costar) and allowed to adhere for 2 h at 37° C. and5% CO₂. 100 μl of Mycobacterium bovis BCG washed three times in freshBMM medium resuspended to a final OD of 0.02 was replaced gently overthe adhered cells and allowed to infect the macrophages for 1 h at 37°C. and 5% CO₂. Free bacteria were removed by three washes and treatmentwith amikacin (200 μg/ml) for 1 h. To initiate the chase, 200 μl freshmedium was added per well and chased for the times indicated. At the endof chase, the medium was removed and the macrophages lysed by additionof 100 μl of incorporation media (7H9 medium with 10% DS supplement,0.15% saponin and 10 μCi/ml of tritiated uracil) to release theintracellular mycobacteria and further incubated for 24 h at 37° C. and5% CO₂. Mycobacteria were lysed by addition of 20 μl of 1N NaOH andincubation at 50° C. for 30 min. Proteins from the lysate wereprecipitated with 80 μl of 50% trichloro-acetic acid and the supernatantharvested using Packard FilterMate Harvester with Unifilter-96, GF/Cfilter. The incorporated counts were measured using the TopCountmicroplate scintillation counter according to the manufacturer'sprotocol. For experiments involving calcineurin inhibitors or calciumchelators, the reagents were added at the time of seeding of the cellsand subsequent steps carried out in the presence of the reagents.

Quantitative Determination of Receptor-Mediated Phagocytosis

To study Fc-receptor mediated phagocytosis 10 μl of a 2% aqueoussuspension of yellow-green fluorescent polystyrene beads (MolecularProbes FluoSpheres beads) were washed twice with PBS and then incubatedfor 1 h at 37° C. on a shaking thermoblock in 1 ml PBS containing 50μg/ml rabbit IgG (Cappel/ICN). After two additional washing steps inPBS, the IgG-opsonized beads were resupended in 3 ml DMEM/10% FCS/2 mMglutamine. For complement receptor-mediated uptake Lactobacillus caseiexpressing GFP was treated at an OD₆₀₀ of 0.2 with 1:10 diluted fresh orheat-inactivated (56° C., 30 min) human serum in PBS for 30 min at 37°C. on a rotator. Serum treated bacilli were then washed twice in anexcess volume of PBS and resuspended in DMEM/10% FCS/2 mM glutamine atan OD₆₀₀ of 2. Bone marrow derived macrophages were seeded to 80%confluency in 24 well cell culture plates (BD Falcon) and shifted to 4°C. followed by the addition of 250 μl per well of the above mentionedsuspensions of fluorescent beads or serum treated Lactobacilli. To allowphagocytosis, cells were then incubated for 30 min at either 37° C. or4° C. (cold control) and subsequently washed five times with 1 ml PBS/5%FCS on ice. Cells were harvested by scraping and subjected to flowcytometry (FACSCalibur, Becton Dickinson) to determine uptake offluorescent cargo in the FL-1 channel. Only living cells as assessed bythe forward- and side-scatter profile were considered for the analysis.Rate of uptake was measured as the increase in fluorescence as expressedby the median fluorescence intensity.

Calcium Fluorimetry

Cells were loaded with the calcium specific fluorophore Fluo3 (MolecularProbes) (2 μM) for 1 h at 37° C. and 5% CO₂ in the presence of 2.5 mMprobenecid. The cells were washed with Ringer's solution without calciumand re-suspended to a final density of 1 million cells per ml. 3 mMCalcium chloride and 2.5 mM probenecid was added and further incubatedfor 2 h at 37° C. and 5% CO₂. After establishment of a base line, cellswere stimulated with the solutions indicated after 50 seconds. Thefluorescence emission was monitored in channel FL-1 and plotted againsttime in seconds.

The invention claimed is:
 1. A method of induction of immunosuppressionfollowing transplantation comprising administering an effective amountof a coronin 1 modulator for immunosuppression to a patient having atransplant, wherein the coronin 1 modulator is selected from the groupconsisting of: an anti-coronin 1 polyclonal antibody, monoclonalantibody or an Fab fragment thereof; a humanized antibody comprising thebinding portion of an anti-coronin 1 monoclonal antibody; a peptidecomprising a coronin 1 peptide selected from (SEQ ID NO: 1)KSRELRVNRGLDTGRRR, (SEQ ID NO: 2) VSRLEEEMRKLQATVQELQKRLDRLEETVQAK,(SEQ ID NO: 3) MSRQVVRSSKFRHVFGQPAKADQCYE, (SEQ ID NO: 4)VCGHTAPVLDIAWCPHNDNVIASGSEDCTVMVWE, (SEQ ID NO: 5)LEGHTKRVGIVAWHTTAQNVLLSAGCDNVIMVWD, (SEQ ID NO: 6)PEVHPDTIYSVDWSRDGGLICTSCRDKRVRIIE, (SEQ ID NO: 7)DRPHEGTRPVRAVFVSEGKILTTGFSRMSERQVALWD, and (SEQ ID NO: 8)PLSLQELDTSSGVLLPFFDPDTNIVYLCGKGDSSIRYF;

a phospho-peptide comprising a peptide of the sequence of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, or 8, wherein one to five serine and/or threonineresidues are phosphorylated; and an RNA selected from (SEQ ID NO: 9)GUUCCGCCACGUGUUUGGA, (SEQ ID NO: 10) UCCAAACACGUGGCGGAAC,(SEQ ID NO: 11) GGCCGACCAGUGCUAUGAA, (SEQ ID NO: 12)UUCAUAGCACUGGUCGGCC, (SEQ ID NO: 13) GAUGUGCGCGUCUCACAGA,(SEQ ID NO: 14) UCUGUGAGACGCGCACAUC, (SEQ ID NO: 15)CCCUAAGUUUGUGGCCCUG, (SEQ ID NO: 16) CAGGGCCACAAACUUAGGG,(SEQ ID NO: 17) GUUUGUGGCCCUGAUCUGU, and (SEQ ID NO: 18)ACAGAUCAGGGCCACAAAC.


2. The method according to claim 1, wherein the coronin 1 modulator isan anti-coronin 1 polyclonal or monoclonal antibody, or an Fab fragmentthereof.
 3. The method according to claim 1, wherein the coronin 1modulator is an anti-coronin 1 Fab fragment.
 4. The method according toclaim 1, wherein the coronin 1 modulator comprises a peptide of SEQ IDNO: 1, 2, 3, 4, 5, 6, 7 or
 8. 5. The method according to claim 1,wherein the coronin 1 modulator is a phospho-peptide comprising apeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, wherein one to fiveserine and/or threonine residues are phosphorylated.
 6. The methodaccording to claim 1, wherein the coronin 1 modulator comprises an RNAof the sequence (SEQ ID NO: 9) GUUCCGCCACGUGUUUGGA, (SEQ ID NO: 10)UCCAAACACGUGGCGGAAC, (SEQ ID NO: 11) GGCCGACCAGUGCUAUGAA,(SEQ ID NO: 12) UUCAUAGCACUGGUCGGCC, (SEQ ID NO: 13)GAUGUGCGCGUCUCACAGA, (SEQ ID NO: 14) UCUGUGAGACGCGCACAUC,(SEQ ID NO: 15) CCCUAAGUUUGUGGCCCUG, (SEQ ID NO: 16)CAGGGCCACAAACUUAGGG, (SEQ ID NO: 17) GUUUGUGGCCCUGAUCUGU, or(SEQ ID NO: 18) ACAGAUCAGGGCCACAAAC.